A Unique Approach to Monitor Stress in Coral Exposed to Emerging Pollutants Didier Stien✉, Marcelino Suzuki, Alice M

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OPEN A unique approach to monitor stress in coral exposed to emerging pollutants Didier Stien✉, Marcelino Suzuki, Alice M. S. Rodrigues, Marion Yvin, Fanny Clergeaud, Evane Thorel & Philippe Lebaron

Metabolomic profling of the hexacoral Pocillopora damicornis exposed to solar flters revealed a metabolomic signature of stress in this coral. It was demonstrated that the concentration of the known steroid (3β, 5α, 8α) -5, 8-epidioxyergosta- 6, 24(28) - dien- 3- ol (14) increased in response to octocrylene (OC) and ethylhexyl salicylate (ES) at 50 µg/L. Based on the overall coral response, we hypothesize that steroid 14 mediates coral response to stress. OC also specifcally altered mitochondrial function at this concentration and above, while ES triggered a stress/infammatory response at 300 µg/L and above as witnessed by the signifcant increases in the concentrations of polyunsaturated fatty acids, lysophosphatidylcholines and lysophosphatidylethanolamines. Benzophenone-3 increased the concentration of compound 14 at 2 mg/L, while the concentration of stress marker remained unchanged upon exposition to the other solar flters tested. Also, our results seemed to refute earlier suggestions that platelet-activating factor is involved in the coral infammatory response.

Coral reefs are experiencing an unprecedented planet-wide decline1. Tis decline has been attributed to several anthropogenic factors, including global warming, overfshing, and pollution. Widely used for skin protection against cancer, solar flters are regularly released in the sea from populated coastal zones or in sites dedicated to touristic activities including a bathing zone. Nonetheless, the impacts of solar flters on corals have been relatively understudied to date. An early article from Danovaro and coworkers2 demonstrated that solar flters can induce coral bleaching by promoting viral infections. More recently, it was shown that several UV flters in the benzophenone class, along with octocrylene (OC), exert direct detrimental efects on corals3–7. Additionally, it has been shown that other ingredients in sunscreens and cosmetics exacerbate the toxicity of the UV flters OC and octinoxate8, while Fel et al. reported that many UV flters, including OC have little or no efect on corals9. According to Downs et al., benzophenone-3 (BP3) induced concentration-dependent planulae coral bleaching, DNA-AP lesions, ossifcation of the planula, and planulae mortality, and these adverse efects were exacer- bated by light3. In our previous work, we compared the metabolomic profles of exposed and unexposed corals and demonstrated that OC triggers mitochondrial dysfunction that results in the accumulation of acylcarnitines7. Also of great concern were both the accumulation of OC derivatives in coral tissues and the concentration at which the toxicity was detected. In a 1-week exposure experiment, 19 OC derivatives were found in the coral tissue, and the response-inducing concentration was 50 µg/L, a concentration only 5 to 10 times higher than the highest environmental concentrations reported in the literature10,11. Since wild corals are exposed to solar flters for longer periods of time, it has been hypothesized that OC does impact corals in areas where it is continuously released. Other groups have also described the accumulation of UV flters in coral tissues8,12,13. Tese data further increase the interest of studying coral response to pollutants. On July 2, 2018, and beginning January 1, 2021, Hawaii banned the sale or distribution of sunscreens containing oxybenzone or octinoxate in its territory14. Lately, Palau restricted the sale and use of reef-toxic sunscreens15. In that ruling, reef-toxic sunscreens were BP3, octinoxate and OC, the manufacturing, importation or sale of which will be prohibited in the Republic of Palau afer January 1, 2020. In the current context, where national legislations are evolving to promote more sustainable tourism while little is known on the impact of UV flters on coral, it was key to evaluate more solar flters and to introduce a practical reliable tool to quantify coral responses to pollutants, while considering the public health importance of sunscreens.

Sorbonne Université, CNRS, Laboratoire de Biodiversité et Biotechnologies Microbiennes, USR3579, Observatoire Océanologique, 66650, Banyuls-sur-mer, France. ✉e-mail: [email protected]

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Maximum concentration in fnal producta Abbr. Name Alternative names Cmpd. class CAS # Formula USA EU Aus.

OC Octocrylene Acrylate 6197-30-4 C24H27NO2 10% 10% 10% Methylene bis-benzotriazolyl Bisoctrizole, Tinosorb M, MBBT Benzotriazole 103597-45-1 C H N O n.a. 10% 10% tetramethylbutylphenol Milestab 360 41 50 6 2

BP3 Benzophenone-3 Oxybenzone Phenone 131-57-7 C14H12O3 6% 6% 10%

BM Butyl methoxydibenzoylmethane Avobenzone Phenone 70356-09-1 C20H22O3 3% 5% 5% Diethylamino hydroxybenzoyl hexyl DHHB Uvinul A Plus Phenone 302776-68-7 C H NO n.a. 10% 10% benzoate 24 31 4

ES 2-Ethylhexyl salicylate Octyl salicylate, Octisalate Salicylate 118-60-5 C15H22O3 5% 5% 5%

HS Homosalate Salicylate 118-56-9 C16H22O3 15% 10% 15% bis-Ethylhexyloxyphenol Bemotrizinol, Tinosorb S, BEMT s-Triazine 187393-00-6 C H N O n.a. 10% 10% methoxyphenyl triazine Escalol S 38 49 3 5

DBT Diethylhexyl butamido triazone Iscotrizinol, Uvasorb HEB s-Triazine 154702-15-5 C44H59N7O5 n.a. 10% n.a. Uvinul T150, Octyl ET Ethylhexyl triazone s-Triazine 88122-99-0 C H N O n.a. 5% 5% triazone 48 66 6 6

Table 1. UV flters tested. a USA: United States of America, EU: European Union, Aus.: Australia. n.a.: not approved.

In the current work, we studied the impact of 10 UV-flters on coral Pocillopora damicornis using untar- geted metabolomic analysis in order to contribute to a better understanding of coral stress response to emerging pollutants. Results and discussion UV flters. Te UV flters used in this study are listed in Table 1. All are approved in the European Union as cosmetic ingredients. Five of them are not approved by the FDA for human use but are ofen approved in other countries around the world, including those with signifcant coral reef areas such as France (4th most areas) and Australia (2nd most areas)16. Overall, the compound classes are somewhat diverse, with 5 classes for 10 solar flters.

Compared efect of OC and ES on coral metabolomes. In the current study, P. damicornis nubbins were exposed to ES for 7 days at concentrations of 5, 50, 300 and 1000 µg/L. As with OC previously, the extracts prepared from the coral nubbins exposed to ES were analyzed by UHPLC-ESI+-HRMS2 and compared with control experiment7. First, unlike what happened with OC, ES or ES-analogs do not seem to accumulate in the coral, although both compounds possess a 2-ethylhexyl side chain. Our hypothesis is that the ester group in OC is more stable than in ES. ES would then be degraded by carboxylesterase-mediated ester hydrolysis17 or via the bacterial ortho degradation pathway18,19 while OC is degraded by hydroxylation of the 2-ethylhexyl chain and subsequent grafing of fatty acids. As a result, lipophilic OC analogs accumulate in coral tissues, which might lead to further accumulation by the trophic chain. Te metabolomic profles were compared with those of control corals treated with DMSO only (0.25% v/v). Eighteen compounds are signifcantly upregulated at 1000 µg/L ES, and in some cases are also upregulated at lower ES concentrations (Table 2, Fig. 1). Compounds 1–3 are the polyunsaturated fatty acids eicosapentaenoic, docosahexaenoic and arachidonic acid (AA, Fig. 1). Te structures were determined based on the molecular formulas and examination of fragmentation spectra with MS-Finder. Identifcation was confrmed by comparison of retention times and MS/MS spectra with those of commercially available standards. Tese compounds were signifcantly upregulated at 1 mg/L ES and were not afected at lower concentrations (Fig. 1). Until recently, ω3 long-chain polyunsaturated fatty acids (PUFAs) would have been considered as Symbiodiniaceae20 metabolites. Vertebrates lack the ωx desaturases necessary for PUFA synthesis and it was largely accepted that all animals should lack it too. However, it is now well established that many marine invertebrates, including corals (P. damicornis among them), have acquired ωx desaturases by horizontal gene transfer21–23. In mammals, ω3 PUFAs, and in particular AA, that come from the diet are stored and are involved in the infammatory cascade afer cleavage from phosphatidylcholine by phospholipase A2 (PLA2) and transformation into several derived signaling molecules such as leukotrienes and prostaglandins. In corals, increased production of eicosanoids, linked to an increase in the expression of an allene oxide synthase-lipoxygenase (AOS-LOXa) was shown for the sof (octo)coral Capnella imbricata in response to mechanical injury and thermal stress22,24. In stony (hexa)corals, eicosanoids such as 8-hydroxyeicosatetraenoic acid (8-HETE), (S,5Z,11Z,14Z)-8-hydroxy- 9-oxoicosa-5,11,14-trienoic acid and (5Z,12a,14Z)-9-oxo-prosta-5,10,14-trien-1-oicacid were also observed, both in extracts of whole coral tissue, and afer incubation of 14C-labeled AA with tissue homogenates of Acropora millepora, A. cervicornis and Galaxea fascicularis23. Tese results, combined with previous transcriptomic analysis showing upregulation of enzymes linked to eicosanoid production in corals undergoing black disease or thermal stress25,26, led to the hypothesis that the production of eicosanoids could also be associated with stress in stony corals23. Finally, experiments with host switching using the sea anemone Exaiptasia pallida also show evidences towards increases of eicoisanoids afer colonization with a heterologous Symbiodiniaceae27.

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Cmpd. tR Molecular No.a (min) m/z Adduct formula Cmpd. name CAS # Cmpd. class + 1 ☑ 8.77 303.2319 [M + H] C20H30O2 Eicosapentaenoic acid 10417-94-4 Fatty acid + 2 ☑ 9.19 329.2475 [M + H] C22H33O2 Docosahexaenoic acid 6217-54-5 Fatty acid + 3 ☑ 9.33 305.2475 [M + H] C20H32O2 Arachidonic acid 506-32-1 Fatty acid + 4 7.07 480.3449 [M + H] C24H50NO6P 1-O-(3Z-hexadecenyl)-sn-glycero-3-phosphocholine 339984-37-1 Lysophosphatidyl choline + 5 ☑ 7.25 496.3400 [M + H] C24H50NO7P 1-O-Hexadecanoyl-sn-glycero-3-phosphocholine 17364-16-8 Lysophosphatidyl choline + 6 ☑ 7.54 482.3605 [M + H] C24H52NO6P 1-O-Hexadecyl-sn-glycero-3-phosphocholine 52691-62-0 Lysophosphatidyl choline + 7 ☑ 8.17 524.3710 [M + H] C26H54NO7P 1-O-Octadecanoyl-sn-glycero-3-phosphocholine 19420-57-6 Lysophosphatidyl choline + 8 6.84 502.2929 [M + H] C25H44NO7P 1-O-Arachidonoyl-sn-glycero-3-phosphoethanolamine 652149-09-2 Lysophosphatidyl ethanolamine + 9 ☑ 7.89 482.3242 [M + H] C23H48NO7P 1-O-Octadecanoyl-sn-glycero-3-phosphoethanolamine 69747-55-3 Lysophosphatidyl ethanolamine + 10 7.36 438.2979 [M + H] C21H44NO6P 1-O-Hexadec-1′-enyl-sn-glycero-3-phosphoethanolamine 174062-72-7 Lysophosphatidyl ethanolamine + 11 ☑ 8.20 466.3292 [M + H] C23H48NO6P 1-O-(Z)-Octadec-1′-enyl-sn-glycero-3-phosphoethanolamine 174062-73-8 Lysophosphatidyl ethanolamine + 12 9.01 481.2925 [M + Na] C28H42O5 Unidentifed Steroid + 13 9.07 481.2931 [M + Na] C28H42O5 Unidentifed Steroid b + 14 ☑ 11.02 451.3181 [M + Na] C28H44O3 5α,8α-Epidioxyergosta-6,24(28)-dien-3β-ol 55688-50-1 Steroid + 15 11.63 451.3181 [M + Na] C28H44O3 Unidentifed Steroid + 16 12.30 465.3338 [M + Na] C29H46O3 Unidentifed Steroid 17 13.71 377.3200 Frag?c Unknown Unidentifed Steroid + 18 6.70 562.3739 [M + H] C32H51NO7 Unidentifed unknown

Table 2. List of upregulated metabolites upon exposition to ES. aCheck marks indicate that the structure was confrmed by comparison with a commercial standard. For 14, see b. bStructure confrmed by NMR. cTe molecular ion was not found, reported experimental m/z might correspond to a fragment.

Our blastp analysis of the P. damicornis genome identifed several enzymes linked to the production of eicosanoids, including cytosolic and secreted PLA2, AOS-LOXs, 5-lypooxygenases and a leukotriene A4 hydrolase, confrming previous results in the literature (Supplementary Table S1)23,26. However, other than PUFAs, we could not detect any of the eicosanoids, leukotrienes or prostaglandins in P. damicornis extracts, nor could we identify a leukotriene receptor based on blastp searches using the mouse LTB4R and CYSLTR1 as queries. Te closest seven-transmembrane G-protein coupled receptors in P. damicornis were related to receptors in the alpha and beta groups of rhodopsin family (defned by Fredriksson et al.)28 and not the gamma and delta groups as other known leukotriene receptors. Altogether, these results suggest that ω3 PUFAs concentration increased likely in the context of a stress process triggered by ES. Tis efect was only noticeable at the highest exposition concentration, but the downstream signaling molecules or putative receptors were absent or not measurable in our study. Compounds 4–7 have very similar MS/MS fragmentation patterns with common peaks at m/z + + + 60.0808 ([Me3NH] ), 86.0964 ([Me3N-CH = CH2] ), 104.1070 ([Me3NCH2CH2OH] ), 124.9998 + + ([H2O3PO-CH = CH2] ), and 184.0733 ([Me3NCH2CH2OPO3H2] ), showing evidence of the presence of a phosphocholine subunit (Supplementary Scheme S1). Te molecular formula completed the structures as those of lysophosphatidylcholines (LPCs). In the collision-induced dissociation of sodiated 5 (Supplementary Fig. S14), the 5:1 peak intensity ratio of product ions at m/z 104 and 147 indicated that 5 was an sn-1-LPC regioisomer29. Te identifcation of compounds 5–7 was fnally unambiguously established by comparing the retention times and MS/MS spectra with those of commercially available standards. As for ω3 PUFAs, these compounds were significantly upregulated only at 1 mg/L ES (Fig. 1). LPCs are widespread in the animal kingdom30. In mammals, LPCs are proinfammatory lipids upregulated in an event of infammation, inducing pro-infammatory cytokine secretion and activating B lymphocytes31–33. LPCs stimulate time- and concentration-dependent release of arachidonic acid (compound 3) in human coronary artery smooth muscle cells33. Interestingly, it has been proposed that the coral immune system is similar to that of higher organisms, including mammals34–37. In Porites sp., platelet-activating factor (PAF) concentration has been reported to increase during interactions with Acropora cervicornis30 as has the expression of a gene coding for a putative LPC acetyltransferase, the protein responsible for converting LPC to PAF, in response to stress and infammation in mammals38. PAF was not detected in our study, even though the genome of P. damicornis codes for an acyl transferase with high homology to the mammalian LPC acyltransferase, containing the motif HXXXXD responsible for acyltransferase activity, and a putative acyl-acceptor binding pocket domain (Supplementary Table S1). Remarkably, compound 7 is an isomer of PAF and could easily be mistaken for PAF. In our study, its structure was confrmed by comparison with standards of 7 and PAF (Supplementary Figs. S2 and S3). Te fragmentation product at m/z 104 from [7 + H]+ further confrms the identifcation of 7 as the 1-O-octadecanoyl-sn-glycero- 3-phosphocholine39,40. In addition, unlike what was reported for Acropora digitifera, we did not identify a gene coding PAF receptor based on blastp searches using the human PTAFR receptor as a query (Supplementary Table S1). Te closest seven-transmembrane G-protein-coupled receptors in P. damicornis were related to chole- cystokinin receptors in beta groups of the rhodopsin family (defned by Fredriksson et al.)28 and not in the delta group as the PTAFR receptor. Finally, our results show that the expression of LPCs fuctuates widely between

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Figure 1. Identifed upregulated metabolites and absolute integration values for biomarkers 1–18 ion peaks. Te X-axis is in Log scale. For each ion, the lefmost point represents the minimal value, and the rightmost point the maximal one. Te rectangular box represents the 25% quantile to 75% quantile ranges. Te dark line shows the average of the distribution. Signifcance levels relative to DMSO were determined by an ANOVA followed by a Tukey HSD test. Te diferences were not signifcant unless otherwise stated. ***p < 0.001, **p < 0.01, *p < 0.05.

replicates (Fig. 1) and, therefore, cannot be considered as good stress indicators. Te signal was weakly signifcant (p < 0.05 for all 4 LPCs) at 1000 µg/L ES and was not signifcant at lower concentrations. Compounds 8–11 were identifed as lysophosphatidylethanolamines (LPEs) based on molecular formulas, MS/MS fragmentation patterns, and structures proposed by CD for compounds 8–10. Compounds 8 and 9 had very similar fragmentation patterns. For example, 8 is characterized by a series of products resulting from the successive loss of water (m/z 464.3134), ethanolamine (m/z 421.2731), and HPO3 (m/z 341.3045, 100%) (Fig. 2, Supplementary Scheme S2). Te ion at m/z 310.3095 (330.2775 for 9) corresponds to a product formed by transposition of the acyl unit to the amino group followed by β-elimination of the phosphate ester. Tis transposition only occurs for the sn-1-LPE regioisomers41. Te structure of compound 9 was unambiguously confrmed by comparison with a commercial standard. Te concentration of compounds 8 and 9 increased signifcantly at 300 µg/L ES and above (Fig. 1). Compounds 10 and 11 are close analogs as demonstrated by their very similar

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Figure 2. Collision-induced MS/MS spectrum of the compound 9 pseudomolecular ion and identifcation of the key fragment ions.

Figure 3. Collision-induced MS/MS spectrum of the compound 11 pseudomolecular ion and identifcation of the key fragment ions.

MS2 fragmentation patterns, in which the migration of the enol ether chain on the amino group leads to the major fragmentation products (Fig. 3, Supplementary Scheme S3). Te identifcation of compound 10 was confrmed by comparison with a commercially available standard. Compounds 10 and 11 were signifcantly upregulated at 1000 µg/L ES. It has been well established that LPEs and LPCs are produced by hydrolysis of phosphatidylethanolamines and phosphatidylcholines, releasing arachidonic acid or other fatty acids linked to the sn-2 position. Tis reac- tion is usually mediated by a phospholipase A2. An increased phospholipase A2 activity in coral exposed to ES would account for the observed concomitant increase of ω3 PUFAs (1–3), LPCs (4–7) and LPEs (8–11). As dis- cussed above P. damicornis genome mining revealed that the genome of this species codes for a number of secretory (sPLA2) and cytosolic PLA2 (cPLA2) presumably involved in this mechanism. Similar to in mammals42,43, sPLA2 upregulation in coral is visibly associated with the activity of the innate immune system and/or a stress response26,38. Increases in PLA2 expression and arachidonic acid was also shown afer host switching with a heterologous Symbiodiniaceae with sea anemone Exaiptasia pallida even though that study did not see diferences in LPEs and LPCs27. Overall, the metabolomic response of coral afer exposition to ES resembles the signature of a stress/infammatory process triggered by ES. To the exception of compound 14, compounds 12–17 (Fig. 1) were detected with somewhat small response peaks, and it was not always possible to obtain MS/MS spectra. Compounds 12–17 seemed related based on their molecular formulas and on their ionization and fragmentation patterns. Interestingly, the diferential analysis

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showed that all these compounds were signifcantly upregulated when the coral was exposed to ES at concentrations ranging from 50 to 1000 µg/L. It turned out that the concentration of 14 also increased upon exposition to OC7, and at this point, it was clearly necessary to determine unambiguously the structure of compound 14. Extensive fractionation allowed for the isolation of 14 in its pure form, which was ultimately identifed by NMR as the known steroid (3β,5α,8α)-5, 8-epidioxy-ergosta-6,24(28)-dien-3-ol. Compound 14 along with many epidioxy sterol analogs have been described essentially in marine invertebrates, including several cnidarians44–47. Te role and fate of this compound in invertebrates remains an unresolved question. However, steroids are present in the whole animal kingdom. Tey regulate life cycles, mating, and development. In Cnidarians, bioregulatory pathways and hormonal-like signaling remain largely uncharacterized48–50. Nonetheless, a family of nuclear receptors has been found to bind the ancient hormone paraestrol A50,51. According to Khalturin et al., Cnidarian steroids can be transported in the digestive tract and through the mesoglea, and may be involved in intercellular commu- nication. Here, compound 14 concentration increased in coral exposed to pollutants, and these pollutants even- tually triggered a stress response witnessed by the increased production of specifc lipids. We hypothesize that the increased concentration of this class of steroids – and in particular the major compound 14 – is a signal triggering a coral infammatory-like response. Owing to the very small standard deviation within replicates (Fig. 2), 14 could be considered as a good marker of stressed corals. In our experiments, the concentration of 14 signifcantly increased with both ES and OC at 50 µg/L, indicating that both solar flters had a negative impact on coral. However, ES triggered a stress/infammatory response while OC also specifcally altered mitochondrial function7. Last, it should be mentioned that the relative concentration of fve metabolites decreased when the coral was exposed to ES at 1 mg/L (Supplementary Figs. S1, S51–S61). Tese metabolites have been identifed as four mono- galactosyl diacylglycerols (MGDGs 19–22) and one cerebroside (23). In MS2, MGDG fragmentation pattern shows the length and number of unsaturation of each acyl chain. Te relative position of the acyl chains can be assigned based on the relative peak intensities of the sodium adduct fragmentation products. Te peak intensity of the product ion resulting from the loss of the sn-1 fatty acid is always higher than the one resulting from the loss of the sn-2 fatty acid52. Although cerebrosides are widely distributed in the eukaryotes, galactolipids including MGDGs are the main components of plant chloroplast membrane lipids53,54. Decreased MGDGs concentration might point towards an efect of the UV flter on the coral symbiont. It is possible that the coral had begun to bleach although bleaching was not visible to the naked eye. However, specifc toxicity mechanism unbalancing Symbiodiniaceae metabolism or any process disrupting algal symbiont metabolite translocation cannot be ruled out.

Efect of BEMT, BM, BP3, DBT, DHHB, ET, HS, and MBBT. Te efect of 8 other solar flters was examined based on the comparison of global metabolomic profles between exposed and unexposed coral, but it was also examined in light of the putative increased concentration of compound 14 in exposed corals. In general, the frst step consisted of exposing coral at 1000 µg/L of each solar flter and testing lower concentrations when an efect was detected. BP3 was tested at 2000 µg/L as well because very high environmental concentrations of this compound have been occasionally detected3. Te relative variations in compound 14 concentration are reported in Fig. 4. We observed that BEMT, DBT, DHHB, ET and MBBT do not seem to afect the overall coral metabolome at the highest concentration tested. Specifc measurement of the concentration of compound 14 in treated ver- sus untreated coral confrmed the apparent innocuousness of these fve molecules, as the concentration of 14 remained stable. It was found that the concentration of 14 increased at 2 mg/L BP3, indicating that BP3 most certainly afects wild coral at the highest published environmental concentration3. Coral exposure to BM does not induce an increase in the concentration of compound 14. Nevertheless, the presence of undetermined ions in the global coral metabolome will require further investigation. Last, HS does not alter either the concentration of compound 14 or the overall metabolome of P. damicornis. However, polyps of coral exposed to HS at 1 mg/L were closed at the end of the assay, while those of control corals were not, as if the coral reacted although its metabolome was not signifcantly altered (Supplementary Fig. S61). Conclusion Tis work establishes a metabolomic signature of stressed P. damicornis. In this coral, the known steroid (3β, 5α,8α)-5,8-epidioxy-ergosta-6,24(28)-dien-3-ol (14) is viewed as a steroid hormone that could trigger coral response to pollutants. OC was the most toxic of the tested UV flters. It induced coral stress response, while triggering mitochondrial dysfunction at 50 µg/L. Of concern was also the previously reported accumulation potential of possibly toxic coral-modifed OC derivatives. ES comes second in terms of toxicity. ES triggered coral stress response at 50 µg/L, inducing a signifcant increase in the concentration of compound 14. At 300 µg/L ES and above, the relative concentration of several PUFAs, LPCs and LPAs also increased. ES may also have induced par- tial coral bleaching at 1 mg/L although this remains to be frmly established. BP3 was also toxic at 2 mg/L in our assay. BEMT, BM, DBT, DHHB, ET, HS and MBBT did not afect coral metabolism at any of the concentrations tested, although in the case of HS and BM, further investigations are needed to evaluate their potential efects. More investigations are also needed to clarify the role of compound 14 in coral hormonal response to pollutants. Methods General experimental procedures. Nuclear Magnetic Resonance (NMR) spectra were recorded on a JEOL ECZ500R spectrometer equipped with a 5-mm inverse detection FG/RO Digital Autotune Probe. Chemical shifs (δ) are reported as ppm downfeld from tetramethylsilane, and the coupling constants (J) are reported in Hertz. High-resolution MS/MS analyses were conducted with a Termo UHPLC-HRMS system7. Analyses were performed in the electrospray positive ionization mode in the range of 133.4–2000 Da in the centroid mode. Te mass detector was an Orbitrap MS/MS FT Q-Exactive focus mass spectrometer. Te analyses were conducted in

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Figure 4. Relative integration values for compound 14 ion peak in exposed coral compared to control animals. Te vertical dashed line illustrates the 1:1 ratio. Te rectangular box represents the min to max ranges. Te dark line shows the average of the distribution. Signifcance levels relative to DMSO were determined by an ANOVA followed by a Tukey HSD test. Te diferences were not signifcant unless otherwise stated. ***p < 0.001, **p < 0.01, *p < 0.05. Te multiple data points for same concentrations are for repeat experiments.

FullMS-data dependent MS2 mode. In FullMS, the resolution was set to 70,000, and the AGC target was 3.106. In MS2, the resolution was 17,500, AGC target 105, isolation window 0.4 Da, and stepped normalized collision energy 15/30/45 was used, with 15 s dynamic exclusion. Te lock mass option was set for an ion at m/z 144.98215, + corresponding to Cu(CH3CN)2 . For coral profling and comparison of coral profles with standards, the UHPLC column was a Phenomenex Luna Omega polar C-18 150 × 2.1 mm, 1.6 µm. Te column temperature was set to 42 °C, and the fow rate was 0.5 mL.min−1. Te solvent system was a mixture of water (solution A) with increasing proportions of acetonitrile (solution B), and both solvents were modifed with 0.1% formic acid. Te gradient was as follows: 2% B 3 min before injection; then from 1 to 13 min, there was a shark fn gradient increase of B up to 100% (curve 2), followed by 100% B for 5 min. Te fow was diverted (not injected into the mass spectrometer) before injection, up to 1 min afer injection. For fast analysis of the fractions of coral extract, the UHPLC column was a Termo Scientifc Accucore Vanquish C18 + 50 × 2.1 mm, 1.5 µm. Te column temperature was set to 42 °C, and the fow rate was 0.5 mL.min−1. Te gradient was as follows: 2% B 1 min before injection; then from 0 to 4 min, there was a shark fn gradient increase of B up to 100% (curve 2), followed by 100% B for 1 min. Te fash chromatography was performed with a Teledyne ISCO CombiFlash Companion equipped with a Büchi FlashPure Ecofex C18 50 µm 40 g column. Te column was equilibrated with water:CH3CN 4:6. Te fow rate was 40 mL/ min. Te injection was performed in the solid phase, with fraction F3 impregnated on C18 silica (1 mL). Te gradient was 60% acetonitrile until 1 min afer injection; then, from 1 to 20 min, there was a linear gradient increase of acetonitrile up to 100%, followed by 100% acetonitrile for 10 min. Efuents were collected in 30 s fractions. Te preparative HPLC was conducted with a hybrid system mounted with a Reodyne manual injector, 2 Varian PrepStar 218 HPLC pumps, a Dionex Ultimate 3000 variable wavelength detector, and a Dionex Ultimate 3000 fraction collector. Te column was a Phenomenex Luna C18 250 × 21.20 mm, 5 µm. Te gradient was as follows:

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90% acetonitrile from 0 to 8 min, 91% from 8 to 16 min, 92% from 16 to 32 min, and 100% for 13 min. Te fow rate was 20 mL/min, and fractions were collected every 30 s. Te second preparative HPLC was performed on small scale with a Dionex ultimate 3000 system equipped with a deaerator, an HPG-3200SD pumping device, an autosampler, a column oven, a diode array detector and a fraction collector. Te column was a Phenomenex Luna C18 150 × 4.60 mm, 5 µm. Te solvent was an isocratic mixture of water:CH3CN 15:85 modifed with 0.1% formic acid. Te fow rate was 1 mL/min, and fractions were collected every 30 s.

Chemicals. Te solar flters used in this study are listed in Table 1. BEMT, BM, BP3, and MBBT were purchased from Sigma-Aldrich, Saint-Quentin Fallavier, France. DBT, DHHB, ES, ET, HS, and OC were provided by Pierre Fabre Laboratories. Standards of platelet-activating factor (PAF) and compounds 1–3, 5–7, 9, and 11 were purchased from AnalyticLab, Montpellier, France.

Pocillopora damicornis. Fragments of the coral P. damicornis were collected in Oman in 2014 (CITES per- mit 37/2014). Tis procedure had no impact on the wild population as 1–5% of few coral colonies were collected. Te coral was acclimated in tanks at the Banyuls Oceanological Observatory. New colonies were obtained in the laboratory from the fragments and used for our experiments. Te corals were maintained in artifcial sea water (ASW) prepared with reverse osmosis purifed water and Reef Salt SeaChem salts. Salinity was adjusted to 36 g/L, pH = 8, and the temperature was set at 24 °C. All experiments were conducted with the same ASW.

Exposition of coral to solar flters, extraction and metabolomic analyses. Te coral exposition protocol, the extraction, the metabolomic profling and the statistical analyses were conducted as described before for OC7. Tey are also reported in Supplementary Information. Five replicates were used for each condi- tion, and in some cases, the conditions were repeated. Te standard compounds were diluted in MeOH (≈ 10 µg/ mL each, 1 µL injected) and were analyzed in the same conditions as the coral extracts for comparison of retention times, MS and MS/MS spectra. All standards were identical to the coral metabolites in retention times and MS/ MS spectra (see supplementary information). Interpretations of the metabolomic data and MS/MS spectra were conducted with the help of Compound Discoverer 2.1 and FreeStyle 1.3 (Termo Fisher Scientifc, Villebon, France), and MS-Finder 3.0455,56. Te same procedure as in Stien et al. (2019) was used for Compound Discover. Extracted ion chromatograms for compounds 1–18 along with ESI+-HRMS spectra, collision-induced dissociation spectra and comparison with commercial standards are provided in Supporting Information (Supplementary Figs. S4–S50).

Isolation and characterization of compound 14. Approximately 200 2–5 cm-long coral nubbins were cut from the branch tips of mother colonies. Te nubbins were placed in a large Erlenmeyer fask and covered with MeOH:CH3CN 1:1. Afer 24 h at room temperature, the fask was sonicated for 20 min, the coral pieces were removed by fltration, and the solvent was evaporated to give the crude coral extract (2.5 g). Te crude extract was dissolved in the smallest amount DMSO possible and was purifed by SPE with a phenomenex Strata C-18 150 mL column. Te column was equilibrated successively with CH3CN and water. Elution was performed with water (300 mL, F1), with water:CH3CN 1:1 (300 mL, F2) and fnally with MeOH:CH2Cl2 8:2 (300 mL, F3). Fractions were evaporated and diluted in MeOH at 1 mg/mL for LC/MS analyses (1 µL injected). Te target compound at tR ≈ 11.02 min and m/z 395.3308 was detected in fraction F3 (0.29 g). Fraction F3 was purifed by fash chromatography, providing 80 fractions. Te analysis of the fractions demonstrated that the target compound was detected in fractions 45 to 63. Tese fractions were gathered and evaporated. For injection, fraction F3.45–63 (49.95 mg) was diluted in water:CH3CN 1:9 (2 mL). Preparative HPLC was performed twice with 1 mL injected. Te targeted compound was concentrated in fractions 40 to 45, which were gathered and evaporated. Te resulting fraction F3.45-63.40-45 (5.91 mg) was diluted in water:CH3CN 15:85 (1.5 mL) and was purifed by small-scale preparative HPLC by portions of 250 µL (6 injections). Te desired compound 14 was isolated in pure form from fractions 27–29 (0.9 mg).

Analytical data for compound 14. 5α,8α-Epidioxyergosta-6,24(28)-dien-3β-ol (14). 1H NMR (500 MHz, CDCl3): δ 0.81 (s, 3 H, H-18), 0.88 (s, 3 H, H-19), 0.94 (d, 3 H, J = 6.5 Hz, H-21), 1.02 (d, 3 H, J = 6.8 Hz, H-26), 1.03 (d, 3 H, J = 6.8 Hz, H-27), 1.21 (m, 1 H, H-17), 1.16 (m, 1 H, H-22a), 1.22 (m, 1 H, H-11a), 1.23 (m, 1 H, H-12a), 1.39 (m, 1 H, H-16a), 1.408 (m, 1 H, H-15a), 1.411 (m, 1 H, H-20), 1.22 (m, 1 H, H-11a), 1.496 (m, 1 H, H-9), 1.497 (m, 1 H, H-11b), 1.53 (m, 1 H, H-2a), 1.54 (m, 1 H, H-22b), 1.55 (m, 1 H, H-14), 1.63 (m, 1 H, H-15b), 1.69 (dt, 1 H, J = 13.5, 3.4 Hz, H-1a), 1.85 (m, 1 H, H-2b), 1.89 (m, 1 H, H-23a), 1.94 (m, 1 H, H-16b), 1.95 (m, 1 H, H-1b), 1.98 (m, 1 H, H-12b), 1.91 (dd, 1 H, J = 13.9, 11.7 Hz, H-4a), 2.09 (brdd, 1 H, J = 10.9, 4.7 Hz, H-23b), 2.11 (ddd, 1 H, J = 13.9, 4.9, 1.9 Hz, H-4b), 2.22 (heptd, 1 H, J = 6.8, 1.0 Hz, H-25), 3.97 (m, 1 H, H-3), 4.65 (brq, 1 H, J = 1.4 Hz, H-28a), 4.72 (brs, 1 H, H-28b), 6.24 (d, 1 H, J = 8.5 Hz, H-6), 6.51 (d, 1 H, J = 8.5 Hz, H-7); 13C NMR (125 MHz, CDCl3): δ 12.6 (C-18), 18.2 (C-19), 18.6 (C-21), 20.6 (C-15), 21.8 (C-27), 22.0 (C-26), 23.4 (C-11), 28.2 (C-16), 30.1 (C-2), 30.9 (C-23), 33.8 (C-25), 34.4 (C-22), 34.7 (C-1), 35.2 (C-20), 36.9 (C10), 37.0 (C-4), 39.4 (C-12), 44.7 (C-13), 51.1 (C-6), 51.6 (C-14), 56.3 (C-17), 66.5 (C-3), 106.1 (C-28), 130.7 (C-7), 135.4 (C-6), + + + + 156.6 (C-24); HR-ESI -MS m/z found 429.3362 [M + H] ; calcd. for [C28H45O3] : 429.3363; HR-ESI -MS m/z + + + 395.3307 (100, [M + H-H2O2] ), 429.3361 (41, [M + H] ), 377.3202 (41, [M + H-H2O2-H20] ), 411.3255 (24, + + + 2 + [M + H-H20] ), 451.3180 (8, [M + Na] ); HR-ESI -MS for [M + H] ion m/z 429.3357 (100), 81.0698 (45), 411.3251 (43), 95.0854 (33), 107.0853 (22), 69.0698 (21), 109.1011 (20).

P. damicornis genome mining. To interpret the possible roles of the identifed stress biomarkers, we que- ried a number of possible putative enzymes and receptors leading to the synthesis, modifcation and binding of diferent phospholipids, polyunsaturated fatty acids and oxylipins against proteins coded by the P. damicornis

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genome [NCBI genome 22550, ASM380409v1 reference annotation release 100/annotated proteins36] using the web-based blastp program of the NCBI and default parameters. Reciprocal blastp searches were also conducted using the web-based tool against the UniProt/Swiss-Prot, and model organism (landmark) databases, and in some specifc cases, the conserved domains (CDD) search. Query sequences for putative enzymes were retrieved by text searches in the MetaCyc, Brenda and KEGG databases, and whenever possible, the closest (based on phylogenetic proximity) experimentally validated sequences were used. Receptor sequences for queries were identifed by text searches in the GLASS-GPCR (https://zhanglab.ccmb.med.umich.edu/GLASS/index.html) database using the same selection criterion as for the enzymes.

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Acknowledgements We thank the BIO2MAR platform (http://bio2mar.obs-banyuls.fr) for providing technical support and access to instrumentation. We thank the Pierre Fabre Laboratories for fnancial sponsorship to the laboratory, and the European Marine Biological Resource Center (EMBRC) at the Observatoire Océanologique de Banyuls, France, for providing access to the Banyuls aquarium facilities. Finally, the authors would like to thank Rémi Pillot and Pascal Romans (Sorbonne Université) for their help in developing the coral assay. Author contributions D.S. and P.L. conceived the work. A.M.S.R. and M.Y. isolated compound 14. F.C. and E.T. performed coral exposition to UV flters. D.S. conducted metabolomic profling, analyzed LC-MS and NMR data, and prepared the manuscript including fgures and tables. M.S. performed P. damicornis genome mining and amended the manuscript accordingly. All authors corrected the manuscript and have given approval to the fnal version. Competing interests Te work reported in this article was fnanced in the context of the Pierre Fabre Skin Protect Ocean Respect action. It was neither supervised nor audited by Pierre Fabre Laboratories. Te interpretation and views expressed in this manuscript are not those of the company.

Additional information Supplementary information is available for this paper at https://doi.org/10.1038/s41598-020-66117-3. Correspondence and requests for materials should be addressed to D.S. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afliations.

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